TMEM16 Structure Published

The structure for a member of the TMEM16 family has been resolved by Raimund Dutzler and colleagues (pdb id: 4WIS). We are interested in the TMEM16 family because TMEM16a conducts the Ca2+ activated Cl- current immature (and not fertilization-competent) oocytes of Xenopus laevis.

A brief history on this family…

The TMEM16 family was cloned during the sequencing of the human genome. TMEM signifies these genes were predicted to encode for a transmembrane proteins (T for trans, MEM for membrane). The number 16 simply represents the sequence of their cloning (meaning that members of TMEM1-15 families had already been identified – and at lease 51 families were identified after – up to TMEM67!). Based on sequence similarity, 10 genes were placed in the TMEM16 family (TMEM16a-k — there’s no ‘i‘).

Until recently, TMEM16 family members were orphan proteins with no known function. In 2008, three labs (Yang et al. 2008, Rock et al. 2008, and Schroeder et al. 2008) working independently reported that TMEM16a encodes a Ca2+-activated Cl- channel, made a knockout mouse, and began characterization of it’s physiologic role (these channels are important and necessary for survival). Since this initial characterization of TMEM16a, the other members of this gene family have been studied.

So far, only two members of the TMEM16 family have been shown to be Ca2+ activated Cl- channels. By contrast, several other family members are lipid scramblases. Scramblases are proteins that allow for the translocation of phospholipids between sides of a lipid bilayer. Phospholipids are comprised of a fatty acid tail and a negatively charged head group. The negatively charged head groups prevent the phospholipid from freely moving between the intracellular and extracellular side of a biological membrane; scramblases provide a path to allow for such movement.

The first TMEM16 structure

Here, Brunner et al. report the first structure from the TMEM16 family of proteins, and it revealed several interesting features.

TMEM16 protein structure contains two subunits shown. The structure is depicted here in a space filling model, with one subunit in green and the other in blue.

This particular protein was cloned from the fungus Nectria haematococca, functions as a Ca2+ activated scramblase, and does not conduct ions. The lack of ionic permeation is not surprising. Here is a phylogenetic tree that I put together comparing the protein sequences of nhTMEM16 with those of the mouse TMEM16 family members:

Yellow denotes proteins with established ionic permeation, and purple denotes those with established scramblase activities (see also Sukuki et al. 2013. JBC 288: 13305-16). Evidently, nhTMEM16 is more similar to the scramblase TMEM16 proteins, and less similar to the Cl- channels TMEM16a and TMEM16b (tree assembled in Clustal Omega and displayed in Dendroscope).

Even though this particular TMEM16 protein is not a channel, the structure is still quite informative. Here’s a summary of some of what I learned:

nhTMEM16 has 10 transmembrane domains

The nhTMEM16 structure unexpectedly has 10 transmembrane domains. Based on sequence analysis, the TMEM16 family was originally predicted to contain only eight. In fact, when originally characterized in 2008, the TMEM16 family was named anoctamins signifying its ability to conduct anions, and being composed of eight transmembrane domains. Accordingly, their gene names are ANO1-10. Based on this structure, we now have evidence that at least some of the TMEM16 proteins are comprised of ten transmembrane domains.

TMEM16 vs Anoctamins

I think it’s time we stop calling proteins in this family anoctamins. It appears as those a majority of TMEM16 family members are not channels (and therefore do not pass anionic currents), and we now have evidence that these proteins likely include more than eight transmembrane domains. Perhaps better name could include reference to the Ca2+ binding capabilities and dimeric characters of the proteins….

Will TMEM16a and TMEM16b also have 10 transmembrane domains?

It’s not clear that the two Cl- channels in the TMEM16 family will have the same membrane topology as nhTMEM16. Comparing the predicted TMEM16a transmembrane domains (which were computed by hydropathy analysis) and the transmembrane domains resolved in the crystal structure of nhTMEM16, the predictions did a great job finding transmembrane domains that correlate to S1-5 and S10 in the structure. We know that the domains between S5-S10 are important for anion conduction in TMEM16a (see Yang et al., 2008), thus it is likely that we will find substantial differences in this portion of the structure of the TMEM16 channels compared to TMEM16 scramblases. These differences could include fewer domains that completely cross the membrane.

Ca2+ binding site embedded in the transmembrane domain

Another interesting finding includes the resolution of the Ca2+ binding site, which appears to be located in an transmembrane domain: Ca2+ interacts with residues in the S6, S7, and S8 domains. Importantly, the Ca2+ binding residues are highly conserved amongst all TMEM16 family members. The authors demonstrate that mutating these homologous residues in mouse TMEM16a channels reduces their sensitivity to Ca2+. While it is possible that TMEM16a includes a second Ca2+ binding site, is is clear that the channel includes this conserved Ca2+ binding site.

A cartoon ribbon display of the nhTMEM16 structure with the bound Ca2+ ions in red, and the approximate boundaries of the transmembrane domains indicated with two black lines. Note the presence of four Ca2+ ions in the structure; this could be a structural artifact, however, the authors cannot exclude the possibility that each subunit is bound two separate Ca2+ ions.

Some of the residues that interact with Ca2+ in the structure had been identified previously by others (e.g. Tien et al. 2014 Elife), although the location of this Ca2+ binding site within the transmembrane domain was surprising to me. Several lines of evidence indicate that Ca2+ binds TMEM16a channels directly and that calmodulin is not required; both this structure and conservation of the Ca2+ binding residues amongst TMEM16 family members should settle any remaining controversy.

Now that we have a structure of a TMEM16 protein, we can start to ask more specific structural questions about TMEM16a structure and function. Surely there will be structural differences between the scramblases and the channels, but there will also be many similarities (including Ca2+ binding).